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Debugging PCR-based amplification of mutant library
Dear all,
I've come across a rather odd case of PCR problems with a mutant library, which is used in directed evolution experiments. The library was made using site-directed mutagenesis, so that five codons are randomised to all possible amino acids using NNK bases. The library is then subjected to directed evolution and the pool of survivors is analysed by Illumina sequencing. The prep of the sequencing library is simple: I've made primers, which carry the Illumina adapters, random indices, sample indices, and primer binding sites complementary to the mutant library. After running a PCR, I use Invitrogen e-Gels for product size selection, quantify the products using the KAPA kit and put it straight into the Illumina MiSeq sample prep.
The problem at hand is that certain mutants are clearly not coming up in the Illumina reads. To test this, I've taken a mutant for which I've had a suspicion that it's missing from the Illumina amplicon pool and combined it with a well-amplified mutant template. If I run a PCR on this mixed template, ligate it into a TOPO vector, transform it into E. coli to get single colonies and perform Sanger sequencing on preps from various colonies, what I get is either the well-amplified template or a hybrid of the two. The hybrid consists of the first of five randomised codons from the well-amplified mutant and the last four from the other mutant. (If I do the same thing but as a template only use the disappearing mutant I get back the correct sequence.)
In my perspective this problem could appear, because PCR products themselves (here ca. 400bp) act as primers during the PCR, in similar fashion to overlap extension PCR. But that's a mere speculation. I was really wondering if you could suggest any alternative solutions and especially ways to avoid this problem from occurring.
I've tried two different PCR conditions:
1) AccuPrime SuperMix I (to my knowledge frequently used for NGS), annealing temp is 72C.
2) Takara PrimeStar HS, annealing temp is 62C.
The same problem occurs with both.
I'd be really grateful for your help because this problem constitutes a major stepping stone for the project I'm working on.
Thanks!
Simon
I've come across a rather odd case of PCR problems with a mutant library, which is used in directed evolution experiments. The library was made using site-directed mutagenesis, so that five codons are randomised to all possible amino acids using NNK bases. The library is then subjected to directed evolution and the pool of survivors is analysed by Illumina sequencing. The prep of the sequencing library is simple: I've made primers, which carry the Illumina adapters, random indices, sample indices, and primer binding sites complementary to the mutant library. After running a PCR, I use Invitrogen e-Gels for product size selection, quantify the products using the KAPA kit and put it straight into the Illumina MiSeq sample prep.
The problem at hand is that certain mutants are clearly not coming up in the Illumina reads. To test this, I've taken a mutant for which I've had a suspicion that it's missing from the Illumina amplicon pool and combined it with a well-amplified mutant template. If I run a PCR on this mixed template, ligate it into a TOPO vector, transform it into E. coli to get single colonies and perform Sanger sequencing on preps from various colonies, what I get is either the well-amplified template or a hybrid of the two. The hybrid consists of the first of five randomised codons from the well-amplified mutant and the last four from the other mutant. (If I do the same thing but as a template only use the disappearing mutant I get back the correct sequence.)
In my perspective this problem could appear, because PCR products themselves (here ca. 400bp) act as primers during the PCR, in similar fashion to overlap extension PCR. But that's a mere speculation. I was really wondering if you could suggest any alternative solutions and especially ways to avoid this problem from occurring.
I've tried two different PCR conditions:
1) AccuPrime SuperMix I (to my knowledge frequently used for NGS), annealing temp is 72C.
2) Takara PrimeStar HS, annealing temp is 62C.
The same problem occurs with both.
I'd be really grateful for your help because this problem constitutes a major stepping stone for the project I'm working on.
Thanks!
Simon